Because Gram-positive (G+) infections are common and can be life-threatening, it is critical to have a thorough understanding of how these pathogenic bacteria and the various molecules that they release interact with the host immune system. One bacterial component long thought to affect the host inflammatory response is lipoteichoic acid (LTA), a surface component expressed by virtually all G+ bacteria and released into the surrounding environment during bacterial growth and as a result of treatment with some antibiotics. LTA's role in stimulating cells of the innate immune system, particularly monocytes, dendritic cells and macrophages, is supported by a large body of work. In addition, in vitro experiments from our laboratory and others have shown that, while LTA can stimulate cells of the immune system to initiate a pro-inflammatory response, LTA also has the ability to synergize with host macromolecules to greatly enhance this cytokine response. The experiments outlined in this proposal will examine both the in vitro and in vivo interactions of LTA and one of its synergistic partners, hemoglobin (Hb). The specific aims, revised according to the 2-year ARRA funding limit, are to: 1. Determine how LTA synergizes with the host protein Hb. This aim will focus on understanding the mechanisms by which LTA and Hb synergize to result in a 10-fold potentiation of macrophage cytokine secretion and will specifically focus on understanding the role of TLR4 in this process. We will determine the effects of LTA+Hb stimulation on the recruitment of various receptors to the macrophage surface and define which TLR signaling pathways are utilized to transduce these signals. Further understanding of this process will be obtained by looking at the set of TLR-responsive genes that are transcriptionally activated when macrophages are stimulated by LTA+Hb and comparing these data to the genes activated by LTA alone, both in WT and TLR4-/- macrophages. Finally, we will begin to determine what stimulatory molecule is responsible for the TLR4-dependent nature of this response. 2. Determine the consequences of synergistic interactions of LTA and Hb in vivo. We have recently found that Hb potentiates the the IL-6 and TNF-alpha responses of wildtype mice to LTA, indicating that the phenomenon we have studied in vitro is also operative in vivo. We hypothesize that this increased cytokine response will either impede or exacerbate S. pyogenes infections. We will study the release of cytokines into the blood and peritoneal cavities of mice injected i.p. with LTA in the presence and absence of Hb. Lastly, we will determine whether such pretreatments ameliorate or exacerbate a S. pyogenes infection.